1. EDNO regulates vascular tone.
Endothelium-derived nitric oxide (EDNO) is the most potent endogenous vasodilator known, and, by its effect upon vascular resistance and cardiac contractility, is a major regulator of blood pressure (Moncada and Higgs, 1993; Cooke and Dzau, 1997). NO exerts its effects as a vasodilator, in part, by stimulating soluble guanylate cyclase to produce cGMP. A deficiency of EDNO (as in the endothelial NOS knockout, or with administration of NOS antagonists), causes hypertension (Dananberg et al., 1993; Shesely et al., 1996). An overproduction of nitric oxide (NO) (as in sepsis), causes hypotension and cardiovascular collapse (Rees et al.,1990; Petros et al., 1991).
NO is released from the endothelium in response to a wide variety of physiologic stimuli. For over a century physiologists have recognized that as blood flow increases through a conduit vessel, the vessel dilates. This flow-mediated vasodilation is dependent upon the integrity of the endothelium, and is largely due to the release of EDNO in response to endothelial shear stress (Cooke et al., 1990; Cooke et al., 1991a). Endothelial cells also respond to pharmacological stimuli. Most vasoconstrictors, such as norepinepherine, 5-hydroxytryptamine, and angiotensin II, also stimulate NO release by the endothelium (Moncada and Higgs, 1993; Cooke and Dzau, 1997).
In this way the endothelium modulates vascular contractility. These responses have physiological consequences. For example, during exercise or with mental stress, myocardial oxygen demands increase. In normal individuals the epicardial coronary arteries dilate to accommodate the need for increased coronary blood flow. By contrast, individuals with coronary artery disease have a dysfunctional endothelium with reduced EDNO production and/or activity. In these individuals, a paradoxical coronary artery constriction is observed with exercise or mental stress that contributes to reduced coronary blood flow, resulting in myocardial ischeni ia (Cox et al., 1989; Zeiher et al., 1989).
In addition to its role as a vasodilator, EDNO is potent inhibitor of vascular smooth muscle (VSM) proliferation. The proliferation of cultured VSM cells is inhibited by exogenous NO donors and cGMP analogues (Garg and Hassid, 1989). Gene transfer of endothelial NOS into the balloon-injured rat carotid artery in vivo demonstrably increases NO release for days after the transfection, and significantly reduces myointimal hyperplasia due to proliferation of intimal vascular smooth muscle cells (von der Leyen, et al., 1995).
EDNO also affects vascular structure by inhibiting the interaction of circulating blood elements with the vessel wall. Platelet adherence and aggregation is inhibited by EDNO (Radomski et al., 1987; Stamler et al., 1989). The adherence and infiltration of leukocytes into the vessel wall during experimental inflammation is reduced by exogenous administration of NO donors, and is enhanced by administration of NOS antagonists (Lefer et al., 1993; Gxc3xa1boury et al, 1993).
To summarize, in states of vascular injury or inflammation, a deficiency of NO contributes to thrombosis, leukocyte infiltration, and vascular smooth muscle proliferation.
2. The role of NO in atherosclerosis
Atherosclerosis is the major cause of disability in this country and is responsible for 500,000 deaths annually due to coronary artery disease and cerebral vascular attack. Atherosclerosis is accelerated by hyper-cholesterolemia, hypertension, diabetes mellitus, tobacco use, elevated levels of lipoprotein(a) (xe2x80x9cLp(a)xe2x80x9d) and homocysteine. Intriguingly, all of these disorders are characterized in humans by an endothelial vasodilatory dysfunction well before there is any clinical evidence of atherosclerosis (Cooke and Dzau, 1997). In all of these conditions, the abnormality appears to be due in large part to a perturbation of the NOS pathway. In most of these conditions, the abnormality is reversed or ameliorated by the administration of the NO precursor, L-arginine (Cooke and Dzau, 1997). L-arginine is metabolized by NOS to citrulline and NO.
Dr. John Cooke and coworkers were the first to demonstrate that endothelial vasodilator dysfunction could be reversed by administration of the NO precursor. In hypercholesterolemic rabbits, administration of L-arginine normalizes the NO-dependent vasodilation to acetylcholine (Girerd et al., 1990; Cooke et al., 1991b). Subsequently, Dr. Cooke and others have demonstrated that acute administration of L-arginine can reverse endothelial vasodilator dysfunction that is observed in the coronary and peripheral circulation in patients with atherosclerosis, and in subjects at risk for atherosclerosis.
Because NO has inhibitory effects on many of the key processes that promote atherosclerosis (monocyte adherence, platelet aggregation, vascular smooth muscle proliferation), Cooke postulated that chronic enhancement of vascular NO production could inhibit atherogenesis. Indeed, his lab demonstrated that in hypercholesterolemic rabbits, chronic oral administration of L-arginine could enhance vascular NO activity (Cooke et al., 1992; Wang et al., 1994; Tsao et al., 1994). This effect was associated with a striking reduction in vascular lesions. By contrast, administration of NOS antagonists reduced vascular NO synthesis, increased endothelial adhesiveness for monocytes, and accelerated lesion formation (Tsao et al., 1994; Naruse et al, 1994; Cayatte et al, 1994). Cooke and others have shown that EDNO exerts its effects on atherogenesis by suppressing the expression and the signaling of endothelial adhesion molecules such as VCAM-1, and by reducing the expression of chemokines such as monocyte chemotactic protein-1 (Marui et al., 1993; Tsao et al., in press). The inhibition of adhesion signaling by NO appears to be mediated by cGMP, whereas the transcriptional effects of NO appear to be due, in part, to its abrogation of an oxidant-sensitive transcriptional pathway mediated by NF6B (Marui et al., 1993; Tsao et al., in press; Tsao et al., 1995).
Surprisingly, the administration of L-arginine in hypercholesterolemic rabbits with pre-existing lesions not only slows the progression of disease, but actually induces regression of atherosclerosis (Candipan et al., 1996).
Accordingly, enhancement of vascular NO may represent a novel therapeutic strategy for cardiovascular disease. The initial studies in humans are encouraging. Cooke and others have recently demonstrated that chronic oral administration of L-arginine in hypercholesterolemic humans or those with coronary artery disease can enhance vascular NO activity (as assessed by vascular reactivity studies and measurement of urinary nitrogen oxides), inhibit platelet aggregability, and reduce the adhesiveness of peripheral blood mononuclear cells (Bode-Bxc3x6ger et al., 1994; Wolfe et al., 1995; Theilmeier et al., in press; Lerman et al., 1997).
3. ADMA, a deter minant of endothelial dysfunction and novel risk factor for atherosclerosis
ADMA (asymmetric dimethylarginine) is an endogenous antagonist of nitric oxide synthase. Several years ago, Vallance and Moncada demonstrated that, in uremic rats and in patients with renal failure, plasma ADMA levels were elevated 5-10-fold from normal values of about 1 micromolar (Vallance et al., 1992a,b). Plasma from uremic animals and patients (but not controls) induced the constriction of isolated vascular rings. This vasoconstriction was reversed by L-arginine. Moreover, infusions of ADMA into the brachial artery of normal volunteers caused a significant increase in forearm vascular resistance at concentrations of ADMA that are found in patients with renal failure (Vallance et al., 1992b).
Recently, the enzyme that is responsible for degrading ADMA (dimethylarginine dimethylaminohydrolase, or DDAH), has been characterized. An antagonist to DDAH has been developed which blocks ADMA degradation (MacAllister et al., 1996). When the DDAH antagonist is added to vascular rings in vitro, a gradual increase in tone is observed. Again, this vasoconstriction is reversed by L-arginine. These studies suggest that ADMA is continuously being synthesized and degraded. An alteration in the turnover of ADMA can affect NO synthase activity.
Elevated levels of ADMA have been found in patients with hypercholesterolemia and atherosclerosis (Bode-Bxc3x6ger et al., 1996; Yu and Xiong, 1994).
ADMA is formed primarily by methylation of protein arginine inside cells, where it plays an important role in modulating protein-RNA interactions (Liu and Dreyfuss, 1995). Free ADMA is released upon protein turnover, and is probably secreted by most tissues and either passed in the urine or metabolized in the kidney (Tojo et al., 1997). Many types of physiological stress, such as the chronic inflammatory stress associated with atheroma formation, oxidative stress from environmental toxins, and stress which might result from poor nutrition, overweight, or age, is associated with chronic cellular damage and leads to increased rates of protein turnover, which in turn may lead to increased secretion of methylated amino acids and higher circulating levels of these amino acids, including ADMA. Indeed, excretory methylated amino acids have been widely used as markers of protein turnover in, for example, fasting or dystrophic animals (Mizobuchi et al., 1985; Bates et al., 1983).
Virtually all risk factors that are associated with accelerated atherosclerosis are also known to attenuate the synthesis and/or activity of EDNO. As a circulating antagonist of NO biosynthesis, ADMA may be an important determinant of endothelial vasodilator dysfunction, and potentially, an important new risk-factor for atherosclerosis. To further examine the role of ADMA and its importance in cardiovascular disease, methodology must be developed to detect ADMA with greater sensitivity, specificity, and with higher throughput.
Today""s standard assay has many shortcomings. The present day method requires high-performance liquid chromatographic separation of all primary amine-containing components of the plasma after they have been derivatized with a fluorescent label, e.g. o-phthalaldehyde. Reproducibility is notoriously sensitive to column and mobile phase conditions and frequent column cleaning and re-running of standard curves is essential. In addition, parallel runs are not easily performed, limiting the number of determinations one can make. Finally, peak areas must be integrated and compared to standard curves.
An immunoassay for ADMA in bodily fluids would normally be desirable for convenience and speed, however, several obstacles hinder the development of accurate antibody-based assays for ADMA in bodily fluids. Chief among these is that any anti-ADMA antibody must be able to distinguish ADMA quantitatively from four structural analogs, which are present in varying amounts in bodily fluids. These include arginine, symmetric NGNG-dimethyl-L-arginine (SDMA), NG-monomethyl-L-arginine (MMA), and citrulline. Arginine is present in 100-fold excess over ADMA in healthy subjects. SDMA is present in 1-5-fold excess, MMA is present in small amounts, and citrulline is present in 30-fold excess over ADMA. In practice is not realistic to expect high-affinity antibodies to be able to distinguish close analogs by more than a factor of ten or twenty in affinity. However, it is possible to remove most of the arginine and citrulline enzymatically or by solid phase extraction, as described below. SDMA is by far the most troublesome analog as it an isomer of ADMA with identical molecular weight. SDMA and ADMA cannot be separated by solid phase extraction, are very difficult to separate chromatographically, and would be expected to be very difficult for an antibody to distinguish adequately for accurate measurement of ADMA. Since SDMA has a different physiological provenance from ADMA, and is not an inhibitor of NOS, there is no reason to expect SDMA to correlate with ADMA or to correlate with vascular dysfunction. Therefore, it is absolutely essential to distinguish these analogs, and it would be exceedingly difficult if not impossible to do so with an ADMA immunoassay.
DDAH, however, has at least 1000-fold higher activity toward ADMA than SDMA, and is therefore uniquely suited for use in an enzymatic assay for unequivocal measurement of ADMA in complex mixtures. Nevertheless an enzymatic ADMA assay using DDAH is problematical because of a number of interfering components in blood. Importantly, citrulline is present in blood, so that it would provide a large background which would substantially diminish the accuracy of an assay where citrulline is the final product which is determined as a measure of the amount of ADMA in the sample. In addition, urea which is also present in blood cross-reacts in the citrulline assay. Finally, porphyrins in blood interfere with the spectrophotometric determination of citrulline. In order to have an enzymatic assay using DDAH, it is essential to prevent these various blood components from interfering with the assay.
An enzymatic assay protocol and compositions are provided for determining ADMA in a blood sample. The method initially removes blood components which may interfere with the assay, e.g. protein removal and enzyme inhibitors or cross-reactive species, followed by concentrating the sample, combining the concentrated sample in the liquid phase with recombinantly prepared NG,NG-dimethylarginine dimethylaminohydrolase (xe2x80x9cDDAHxe2x80x9d). Citrulline, the enzymatic product, is then determined colorimetrically. The assay has high reproducibility and accuracy as compared to other assays, which are less convenient and inconvenient to automate.